The practice of modern biotechnology relies upon a number of different genetic-engineering techniques in order to enable the expression of heterologous genes in various organisms. The application of biotechnology to cultivated mushrooms was initially hampered until certain experimental genetic-transformation systems had been developed. In consideration of molecular breeding and the potential of using mushrooms as expression hosts, researchers have put substantial effort into the development of genetic-transformation systems for edible mushrooms.
Researchers have attempted to develop a transformation system for commercial mushrooms, such as A. bisporus, for the introduction of novel characteristics. For other fungi, as well as plants, animals, and bacteria, the application of gene transfer technology is quite common and has already resulted in commercial application. However, the absence of an efficient, reproducible, stable transformation system generally applicable in a wild-type background in many fungi has strongly hampered molecular-biological research on such organisms.
In the past, most protocols used in fungal transformation involved electroporation of protoplasts (Chakraborty et al., 1991, Robinson and Sharon, 1999, van de Rhee et al., 1996), treatment of CaCl2, polyethylene glycol (Ogawa et al., 1998, Sato et al., 1998), or restriction enzyme-mediated integration (Hirano et al., 2000, Irie et al., 2003, Sato et al., 1998). Only a few reports demonstrated the transformation of L. edodes (Hirano et al., 2000, Irie et al., 2003, Li et al., 2006, Sato et al., 1998). Since these transformation systems mainly relied on troublesome protoplast preparation, they were not applicable to other edible mushrooms which may not yield sufficient regenerable protoplasts and these transformation events might be inefficient or difficult to reproduce in other laboratories. Agrobacterium tumefaciens-mediated transformation has been routinely used for the genetic modification of a wide range of plant species and also demonstrated the ability to transfer DNA from a prokaryote to filamentous fungi (Chen et al., 2000, Combier et al., 2003, De Groot et al., 1998, Leclerque et al., 2004, Mikosch et al., 2001), nevertheless, this method is not necessarily appropriate for all mushroom species. Other fungi transformation schemes are disclosed in WO95/02691 and WO98/45455.
These have either had no success, or not been reproducible. Despite considerable interest in the development of a transformation scheme, no method is in general use today, due to low efficiency or lack of utility and convenience. Thus, there is a need to develop a highly effective and convenient genetic transformation system for mushroom.